Protothecosis

The Protothekose is an infectious disease that can occur in humans, cattle, dogs and other species and is triggered by green algae of the genus Prototheca. These algae and their relatives from the genus Helicosporidium are within the algae to the effect especially since they can cause infections in organisms that have no immune system disorder. The two most common types are Prototheca wickerhamii and Prototheca zopfii. In humans, the disease was first described in 1964 is mostly triggered by P. wickerhamii. For dogs both species are pathogenic. The algae are found worldwide in effluents and soils; Infections are rare but despite the relatively high infection pressure and may with defects in the immune system related. The therapy is not standardized in the literature various methods with antifungals, surgical excision and disinfection are described.

• 2.4.1 microscopy
• 2.4.2 Microbiology
• 2.4.3 Molecular Biology

• 2.5.1 antibiotic resistance in vitro
• 2.5.2 therapy protocols

• 3.1 Domestic Cattle
• 3.2 Domestic Dog
• 3.3 Other animal species

Prototheca

As Prototheca in 1894 for the first time were isolated, they were still treated as fungi. After their taxonomic status has long been controversial, considering Prototheca today than in the course of evolution by mutations in the unicellular green algae of the genus Chlorella genus formed. But contains the cell wall of Chlorella galactose and galactosamine as these substances do not occur in Prototheca. Chlorella contains chlorophyll and thus photosynthesis, whereas Prototheca contain no chlorophyll and saprotroph itself, ie feed on decaying organic material.

Protothekose in humans

Pathogenesis

Little is known about the pathogenesis of Protothekose. Prototheca generally have a low virulence, and infection remain normally localized. Infection occurs on the one hand by superficial contact with contaminated substances; but more often a contamination of skin lesions seems to be with Prototheca.

More severe forms of Protothekose normally only occur when the immune system malfunctions. In particular, the cellular immunity seems to play an important role: Prototheca are phagocytosed by neutrophils in a healthy organism granulocytes ( PMNs ) and rendered harmless, the presence of IgG and heat-stable opsonin is important for optimal control. However, neither neutropenia due to cancer is still the most cases of AIDS normally lead to an increased risk of Protothekose. It comes from the fact that more severe course of the Protothekose with qualitative and quantitative deficiencies associated in the function of PMNs.

Epidemiology

The Protothekose is a rare infection in man and not normally spread from human to human; an infection is present in the environment rather by Prototheca. Commensal on the body surface living Prototheca can trigger in the presence of predisposing factors, an opportunistic infection: Even in healthy people Prototheca on skin and nails as well as in the respiratory and digestive tract could be detected. In more than half of all clinical cases of Protothekose can be a local or systemic immunodeficiency be detected as favoring circumstance.

Risk factors for Protothekose have a weakened immune system related to the use of corticosteroids or cancers of the hematopoietic system, organ transplants and surgery in general, diabetes mellitus, and alcohol dependence. Even diseases that are treated with immunosuppressive drugs that increase the risk of Protothekose. The majority of patients are older than 30 years; But cases in neonates and children are also available.

Geographically comes Protothekose before on every continent except Antarctica. Above average often it is in the southwestern U.S. and in rural areas in Taiwan. Workers in rice fields, fishermen, farmers, aquarium staff and people who have contact with raw seafood are particularly exposed.

Clinic

Protothekose is divided into three clinical forms: skin lesions ( cutaneous Protothekose ), olecranon bursitis ( inflammation of the bursa at the elbow ) and body widespread or systemic infections. Unusual course forms are also described next, so about as urinary tract infection, vaginitis ( inflammation of the female genital organs ), pneumonia, and meningitis. Skin infections and olecranon bursitis usually become chronic; acute systemic progressive forms are rare and occur only in patients with severely compromised immune systems.

Cutaneous Protothekose

Cutaneous Protothekose (skin form) is the most common occurring human form of the disease and does little more than half of all cases. It may occur in connection with injuries to the skin and / or mucous membranes, but is also independent of prior injury. The symptoms develop slowly and usually do not heal spontaneously. The lesions are usually ulcerated ( ulcer -like), purulent and form scabs, but can also occur in other forms. If a Prothotekose as a complication after surgery on, it can cause nodule formation, synovitis ( joint inflammation ) and chronic exuding wounds come.

It is of a lasting several weeks of incubation. The lesions usually are localized and propagate only in immunocompromised patients further. They are located mainly in exposed places, ie on the extremities and face.

Olecranon bursitis

Caused by Prototheca olecranon bursitis is an inflammation of lying back of the elbow bursa, olecranon bursa subcutanea. The infection is usually a result of perforating ( skin -penetrating ) injuries by which the Prototheca can get into the bursa. The symptoms appear several weeks after the injury and express themselves through a swollen, slightly indurated and painful bursa. It also infections caused by contamination of an existing wound and infection are described without previous penetrating injury.

Systemic Protothekose

Systemic or disseminated Protothekose occurs mainly in immunocompromised patients. Worldwide 23 cases are described; at 21 of these were in the pathogen to P. wickerhamii. Structures are most frequently affected skin and subcutaneous tissue, intestine, peritoneum, blood and spleen. Systemic Protothekose occurs as a complication of cancer, organ transplants or AIDS most. In three of the cases described the Prototheka peritonitis was the result of catheterization. Prototheka sepsis as a complication of a central venous catheter is also described. Frequently found in affected patients due to their immune deficiency next to the Protothekose other infections with opportunistic pathogens.

Diagnosis

Protothekose is usually detected rather late, since they as a differential diagnosis of infections does not enjoy a high priority. Typically, a suspected Protothekose comes on only when patients were treated over a long period unsuccessfully against other pathogens. The diagnosis is usually based on the morphological identification of Prototheca under the microscope, whereby different dyes can be used. The examination can be performed directly with wound exudate and / or tissue samples; in addition, a microbiological culture is recommended. In addition, molecular methods have been described for the diagnosis. Serological studies do not seem to diagnose in humans, however, to be eligible.

Microscopy

Prototheca are spherical to ellipsoid, have a pronounced cell wall and contain several thick-walled spores car. The diameter varies between 8.1 × 24 microns and 10.8 × 26.9 microns; the spherical bumper spores have a diameter of 9 microns to 11 microns. In contrast to yeasts, Prototheca not form buds. They can be dyed only poorly with the HE staining, but with Gridley stain, Grocott - Gomori staining or PAS stain easily dyeable. The Prototheca can be morphologically confused with several fungi, including Blastomyces dermatitidis with, Cryptococcus neoformans, and Pneumocystis jirovecii.

Grocott - Gomori staining

PAS staining

In addition to large amounts of tissue sections Prototheca also a number of pathological reactions can be observed. Possible responses range from a granulomatous inflammation with marked tissue necrosis to the complete absence of inflammatory reactions despite detection of Prototheca. In cutaneous Protothekose the organisms are usually in the mid and papillary dermis. Disseminated Protothekosen result in marked eosinophilic infiltrates and fibrosis of the affected organs.

Microbiology

Microbiological identification of Prototheca is based on the appearance of the colonies, microscopic identification and a plurality of characteristic metabolic properties in culture. Prototheca are relatively undemanding and can be cultivated on various culture media routinely available readily. However, many of the traditions of the fungal culture selective culture media are not suitable for breeding, because that also inhibits the proliferation of the Proto counters contained in these cycloheximide. For breeding of Prototheca suitable media are Sabouraud dextrose agar, blood agar, beef broth and brain-heart agar. Since samples often contain other microorganisms in addition to Prototheca, 5 -fluorocytosine and potassium hydrogen phthalate are added to the selection, which inhibit the growth of most bacteria and fungi. From yeasts Prototheca can be distinguished by the addition of ribostamycin that, but not inhibit the growth of Prototheca of yeasts.

The incubation is carried out at 30 ° C for 72 hours. For slow-growing Prototheca incubation for seven days at 25 ° C may be necessary. The optimum temperature is between 25 and 37 ° C, and colonies are usually visible after 48 hours. Macroscopically, they appear soft, moist, yeast-like, white or slightly yellowish. The organisms grow either aerobically or microaerophilic.

P. and P. zopfii wickerhamii the two most important causative agent of Protothekose can be distinguished, which are described in the following table based on several properties:

Molecular Biology

To diagnose a Protothekose microscopic and microbiological studies are usually sufficient. But in addition molecular biological methods can be used: The identification of P. wickerhamii by fluorescence in situ hybridization (FISH ) using the rRNA possible by corresponding DNA probes, as the cell walls of Prototheca by pretreatment with CTAB for these probes can be made ​​permeable. Also, an identification of P. zopfii by PCR of the rDNA is possible.

Therapy

Resistance profile in vitro

Prototheca display in-vitro some natural resistance to antibiotics and antifungals and can also acquire new resistance during therapy. The natural resistance situation is shown in the following table. For P. zopfii addition the presence of a β -lactamase was detected.

* Tetracycline and amphotericin B show a synergistic effect against Prototheca.

The sensitivity of Prototheca to polyenes and azoles is explained that in their cell membranes, ergosterol is included. A MIC test of Prototheca on resistance is in practice usually not necessary because the thus obtained results correlate only partially with the clinical success of treatment. Resistance tests are therefore recommended only for unsuccessful treatment attempts.

Therapy protocols

There are no standardized treatment recommendations nor consistent clinical therapy results for Protothekose. Also, clinical studies comparing different therapies with each other, are not available. In practice, surgical combined with drug therapy forms. Protothekose does not heal spontaneously, and treatment failure is not uncommon.

Cutaneous Protothekose successful therapies described include a complete surgical excision of the affected area of ​​skin, the topical application of amphotericin B and different azole, the topical application of Amphotericin B in combination with systemic administration of tetracyclines, the systemic administration of amphotericin B with and without a cutting and the systemic administration of tetracyclines. Treatment failures are described for tetracycline, Itroconazol, fluconazole, and ketoconazole fluorocytosine; inconsistent treatment results for the systemic administration of penicillin, griseofulvin and emetine and for local applications, disinfectants such as hydrogen peroxide, chlorhexidine, potassium permanganate, copper sulfate, picric acid, ammonium compounds and potassium iodide. The duration of treatment varies from a few days to several weeks.

When caused by Prototheca olecranon bursitis is the therapy in the surgical removal of the infected bursa. Alternatively, a drainage in combination with the instillation of amphotericin B are drawn into the bursa into consideration. Systemic treatment with itraconazole over two months is also recommended.

For Protothekosen disseminated systemic treatment with amphotericin B, a combination of amphotericin B and doxycycline and fluconazole are described. Peritonitis by Prototheca was treated by doses of amphotericin B directly intraperitoneally. The excision of the original site of infection, or the removal of foreign bodies in combination with systemic administration of medication is recommended as cautious therapy method. The therapy with azoles is viewed critically, since most treatment failures occurred with this group of drugs; the use of amphotericin B therefore appears to be more appropriate. The duration of therapy described varies greatly, ranging from five days to eight months; as an extreme case of a therapy caused by Prototheca meningitis with amphotericin B and azoles is described over six years, but the pathogen could not eliminate.

Protothekosen in veterinary medicine

Domestic Cattle

When Domestic Cattle infections with Prototheca lead to intestinal and udder infections (mastitis ). The Prototheca mastitis occurs worldwide; most cases of infected herds are reported from Germany, the USA and Brazil. The Prototheca Mastitis is a clinically severe inflammation of the udder, which is not medikamentell treatable. The infection is apparently maintained over subclinically diseased shedders in a herd. A renovation of existing buildings can be done on the identification and culling of infected animals. For diagnosis are serological tests for antibodies against Prototheca helpful; the detection of P. zopfii can also be performed by PCR. Prototheca not be safely destroyed by pasteurisation of the milk and therefore represents a potential zoonotic risk questions

Housedog

In domestic dogs, some individual cases with cutaneous, systemic and disseminated Protothekose have been described. The first description was in 1969., There is a predisposition for Collies and female animals. Infections with Prototheca either lead to a skin infection, or more frequently a disseminated Protothekose. In this, the algae penetrate through the mouth or nose into the body and cause an intestinal infection. From there it spread to the eyes, brain and kidneys. The symptoms of the disease include diarrhea, weight loss, weakness, eye inflammation, retinal detachment, loss of coordination, and seizures.

Dogs with acute blindness and diarrhea who develop exudative retinal detachment should be examined for Protothekose. Diagnosis is made by culture or directly by the microscopic identification of algae in a biopsy or in the cerebrospinal fluid, vitreous or urine. The treatment of disseminated Protothekose is difficult, but antifungals have proven to be effective in some cases. The prognosis of cutaneous Protothekose is moderate and depends on the possibility of surgical removal of the affected skin. The prognosis of the disseminated form is bad, possibly also with the fact that the disease is usually diagnosed and treated late.

Other animal species

Protothekose is very rare in the domestic cat. The first case was described in 1976; the affected cat had a fluctuating mass on the hind leg. In the few other cases described, it was skin infections.

In Atlantic salmon in a fish farm, an infection of juvenile fish by Prototheca salmonis is described which clinically manifests itself in a kidney infection and histologically systemic infection could be detected in the.